CRISPR-Cas9 System:one-step Multi-target Gene Editing Technology For Saccharomyces Cerevisiae

With rapid progress in DNA synthesis and sequencing,strain engineering starts to be the rate-limiting step in synthetic biology.Here,we report a gRNA-tRNA array for Clustered Regularly Interspaced Short Palindromic Repeats-Cas9(GTR-CRISPR)for multiplexed engineering of Saccharomyces cerevisiae.Using reported gRNAs shown to be effective,this system enables simultaneous disruption of 8 genes with 87%efficiency.We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast.This approach enables disruption of 6 genes in 3 days with 60%efficiency using reported gRNAs and 23%using un-optimized gRNAs.Moreover,we applied the Lightning GTR-CRISPR to simplify yeast lipid networks,resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes.